To track protein expression, localization, activity, interactions, or conformational changes as components of cellular signaling pathways, biologists need general tools for in vivo site-specific labeling of proteins with fluorophores and other useful probes. Traditional chemical labeling methods, such as cysteine-maleimide conjugation, are too promiscuous for in vivo use, and the most widely used genetic method, fusion to green fluorescent protein (GFP), carries a payload of 238 amino acids and limits detection sensitivity due to GFP's dim fluorescence and tendency to photobleach. We are using protein engineering techniques in combination with small-molecule synthesis to develop reagents for the site-specific labeling of any desired protein in vivo. These reagents should allow the productive study of signaling events via attachment of a wide range of biophysical probes (e.g., fluorophores and photoactivatable crosslinkers) to specific protein targets in living cells.